The cyclin-dependent kinases (CDKs) are heterodimeric complexes composed of a catalytic kinase subunit and a regulatory cyclin subunit, and comprise a family divided into two groups based on their roles in cell cycle progression and transcriptional regulation (Meyerson M. et al.: EMBO J. 11:2909-2917, 1992). Members of the first group comprise core components of the cell cycle machinery, and include cyclin D-dependent kinases 4 and 6, as well as cyclin E-CDK2 complexes, which sequentially phosphorylate the retinoblastoma protein (Rb), to facilitate the G1-S transition (Sherr, C. J.: Cell 79: 551-555, 1994.). Cyclin A-dependent kinases 2 and 1 and cyclin B-CDK1 complexes are required for orderly S-phase progression and the G2-M transition, respectively. CDKs are regulated by positive phosphorylation, directed by CDK-activating kinase (CAK; cyclin H/cdk7/MAT1, Harper, J. W. et al.: Genes Dev. 12: 285-289, 1998), as well as negative phosphorylation events (Morgan, D. O.: Nature 374: 131-134, 1995), and their association with cyclins and endogenous Cip/Kip or INK4 inhibitors (Sherr, C. J. and Roberts, J. M.: Genes Dev. 9: 1149-1163, 1995). In malignant cells, an altered expression of CDKs and their modulators, including overexpression of cyclins and loss of expression of CDK inhibitors, results in deregulated CDK activity, providing a selective growth advantage. In contrast to CDKs governing the transitions between cell cycle phases, transcriptional CDKs, including cyclin H-CDK7, and cyclin T-CDK9 (pTEFb), promote initiation and elongatin of nascent RNA transcripts by phosphorylating the carboxy-terminal domain (CTD) of RNA polymerase II (Meinhart, A. et al.: Gens. Dev. 19: 1401-1415, 2005). Because of their critical role in cell cycle progression and cellular transcription, as well as the association of their activities with apoptotic pathways, the CDKs comprise an attractive set of targets for novel anticancer drug development.
The first reported pharmacological CDK inhibitors (6-dimethylaminopurine and isopentenyladenine) were neither particularly active nor selective. However, they provided the first grasp on inhibitory structures, and constituted the starting point for the search for more potent and selective inhibitors. More than 50 inhibitors have so far been described. Their structures were recently extensively reviewed (Fischer, P. M., Gianella-Borradori, A.: Expert Opin. Investic. Drugs 14: 457-469, 2005). Despite striking chemical diversity, all CDK inhibitors share some common properties: (1) they have low molecular weights (<600); (2) they are flat, hydrophobic heterocycles; (3) they act by competing with ATP for binding in the kinase ATP-binding site; (4) they bind mostly by hydrophobic interactions and hydrogen bonds with the kinase; and (5) the backbone carbonyl and amino side-chains of Leu83 act, respectively, as an H-bond acceptor and an H-bond donor to the inhibitors, whereas the backbone carbonyl of Glu81 often acts as an H-bond acceptor. The atomic interaction between inhibitors and CDKs is extensively described (Hardcastle, I. R. et al.: Annu. Rev. Pharmacol. Toxicol. 42: 325-348, 2002). Interestingly, inhibitors of CDKs that act via mechanisms other than competing with ATP have not been described, despite intensive screening and despite the other obvious possibilities of kinase inhibition (e.g. competition with substrate, interference with cyclin binding, and simulation of the natural protein inhibitors).
CDK inhibitors fall into three categories, pan-CDK inhibitors (e.g., deschloroflavopiridol, flavopiridol, oxindole 16 and oxindole 91), those that inhibit CDK1/2/5 (and possibly CDK9) (e.g., olomoucine, (R)-roscovitine, purvalanol B, aminopurvalanol (NG97), hymenialdisine, indirubin-3′-monoxime, indirubin-5-sulfonate, SU9516 and alsterpaullone), and those that are selective for CDK4/6 (e.g. fascaplysin, PD0183812, and CINK4). Only a limited number of inhibitors selective for a single CDK have been described. This is probably due to the conservation of the amino acids lining the CDK ATP-binding pocket (Shapiro, G. I. J. Clin. Oncol. 24:1770-1783, 2006).
The carboxy-terminal domain (CTD) of RNA polymerase II is regulated by phosphorylation mediated by CDKs. The human RNA polymerase II CTD contains 52 tandem repeats of the consensus heptapeptide sequence. Cyclin T-CDK9 (also so called P-TEFβ) preferentially phosphorylates the Ser2 sites of this sequence to promote transcriptional elongation. It is likely that cyclin T-CDK9 can phosphorylate the Ser5 position as well (Palancade, B., Bensaude, O.: Eur. J. Biochem. 270: 3859-3870, 2003). Cyclin H-CDK7/MAT1, in the complex of transcription factor TFIIH, preferentially phosphorylates Ser5, which facilitates promoter clearance and transcriptional initiation (Meinhart, A. et al.: Gens. Dev. 19: 1401-1415,2005). Cyclin H-CDK7 therefore plays a role both as cell cycle and transcriptional CDK; it acts as both CAK and CTD kinase.
Flavopiridol is the most potent known inhibitor of CDK9 (Chao, S. H., Price, D. H.: J. Biol. Chem. 276: 31793-31799, 2001). Whereas the IC50 values for other CDKs range from 100 to 400 nmol/L with Ki values between 40 and 70 nmol/L, the binding of flavopiridol to the ATP binding site of CDK9 is significantly tighter (K, =3 nmol/L). Therefore, the inhibition by flavopiridol of CDK9, as well as CDK7, has profound effects on cellular transcription (Lu, X. Et al. Mol. Cancer ther. 3:861-872, 2004). Seliciclib® (R-roscovitine) also inhibits cyclin T-CDK9 and cyclin H-CDK7 in addition to cyclin E-CDK2 (McClue, S. J. et al. Int. J. Cancer 102: 463-468, 2002) and affects RNA polymerase II CTD phosphorylation, which is associated with a decrease in MCl-1 as well as other antiapoptotic proteins in CLL cells (Alvi, A. J. et al. Blood 105: 4484-4491, 2005). The proapoptotic activity of both flavopiridol and seliciclib in multiple myeloma cell lines occurs by a similar mechanism (Raje, N. et al. Blood 106: 1042-1047, 2005).
Our current work is focused on development of novel classes of compounds potently targeting cell cycle with variable effects on the transcriptional CDK7 and 9. Some of these inhibitors have already been described and originate from different classes of compounds, i.e. purines (Olomoucine II, Kry{hacek over (s)}tof V. et al. Cell. Mol. Life Sci. 62, 1763-1771, 2005) and arylazopyrazoles (Kry{hacek over (s)}tof, V. et al. J. Med. Chem. 49: 6500-6509, 2006). Based on our present knowledge of inhibitor/CDK7/9 interactions, we designed a second-generation inhibitors acting at concentrations close to the cellular concentrations of the kinases.
The present invention provides a series of novel substituted 6-(2-hydroxybenzylamino)purine derivatives that are useful for inhibition of cyclin-dependent kinase 5, 7 and 9. This group of new purine derivatives is characterised by an unusual CDK inhibitory activity thus bringing not only strong anticancer properties to the compounds but also up to now unknown anti-inflammatory activity. Hence they can be used as antimitotic and apoptotic drugs, particularly as anticancer drugs in treatments of metastatic tumors. It is the aim of this invention to provide new anticancer compounds having improved selectivity and efficiency index, i.e. that are less toxic yet more efficacious than the analogues known heretofore.